The objectives of this proposal are to use isolated myocytes prepared by collagenase digestion of perfused rat and guinea pig hearts to study various aspects of calcium homeostasis and its perturbation under conditions of hormonal stimulation and interactions leading to irreversible tissue injury. The use of Quin 2 and other intracellular Ca2+ indicators will be thoroughly investigated as tools to measure the intracellular free Ca2+ and Ca2+ transients induced by electrical stimulation of the myocytes. Contraction of single cells will be monitored by video-microscopy and compared with the fluorescence changes of intracellular probes using a computerized image analysis system. A dual channel fluorometer for simultaneous measurement of Quin 2-Ca fluorescence and pyridine nucleotide fluorescence will be used with bulk cell suspensions. In addition, permeant and non-penetrating membrane potential-sensitive and pH-sensitive fluorescent probes will be used to monitor continuously the electrical potential across the plasma and mitochondrial membranes and proton gradients. Results will be compared with measurements of adenine nucleotide and creatine phosphate contents assayed by classical sampling techniques to assess the energy state of the cells. The calcium distribution between mitochondria and other vesicular pools will be investigated using rapid cell disruption techniques and by sequential additions of uncoupling agent and the Ca2+ ionophore A23187, with arsenazo III as an extracellular Ca2+ indicator. Calcium transport across the plasma membrane will be monitored by means of a Ca2+-selective electrode and Ca2+ sensitive dyes. Using combinations of the above techniques, emphasis will be given to understanding the relationships between altered energy status of the cell, intracellular free Ca2+, the calcium distribution and plasma membrane integrity during oxygen and substrate deprivation and upon reinitiation of oxidative phosphorylation. Further studies will relate to the mechanism of sarcolemma damage and the relationship between the Ca2+ transient, the source of contractile calcium and the role of mitochondria in the regulation of cytosolic free Ca2+ and the activity of mitochondrial Ca2+-sensitive dehydrogenase. In addition, we will investigate whether inositol trisphosphate, the product of phosphatidylinositol 4,5-bisphosphate breakdown, acts as a Ca2+-mobilizing second messenger in cardiac tissue, and if so its intracellular site and mechanisms of action will be studied. Further studies will utilize 31P and 1H NMR techniques to investigate post-ischemic reperfusion of rat hearts.